An in Vitro Antifungal and Antiaflatoxigenic Properties of Commiphora myrrha and Prunus mahaleb

Aflatoxins and especially aflatoxin B, are the devastating contaminant of food and feed products with hazardous effects to mankind and his domestic animals. These investigations were set to evaluate the effect of various levels of Commiphora myrrha resin (1.0, 1.25, 2.25, and 3.25 g/100 ml) and Prunus mahaleb seed extract (0.75, 1.5, 2.5, and 3.5 g/100 ml) on the growth and aflatoxin secretion by two aflatoxigenic strains of Aspergillus flavus and A. parasiticus. The two plant extracts significantly (p<0.05) decreased aflatoxin secretion, and inhibited the fungal growth. Resin of C. myrrha displayed 51.9-95.7% reduction in total aflatoxin secretion by A. flavus, and 46.9-92% for A. parasiticus, and Seed extract of P. mahaleb decreased aflatoxin up to 53.7-95.8% and 40-94.7%, respectively. The inhibition of aflatoxin B (B1 and B2) by myrrh resin and seed extract of mahaleb ranged between 51.7-93.5, 50-93.6% (A. flavus) and 39.5-89.7%, 37.9-93% (A. parasiticus). The mycelial dry weight of A. flavus and A. parasiticus ws decreased up to 46.1-58.7%, 28.9-51.3% (Myrrh resin), and between 45-56.9%, 33.3-55.9% (Mahaleb seed extract). Nonetheless, the two plant extracts did not detoxify aflatoxin B1. Therefore, it apparent that the resin of C. myrrha and seed extract of P. mahaleb affected the biosynthesis pathway of aflatoxins. Thus, they can be recommended as effective natural plant biopreservative against aflatoxin contamination of food and feed products.

Prunus mahaleb L. (sync. Cerasus mahaleb L. Mill.) of the family Rosacease is known as English cherry, Rock cherry, St. Lucie cherry and "mahleb, mahaleb, mahlab" in Arabic which grown abundantly in West Asia, North Africa, Middle East, and sometimes found in Eastern and Central Europe (Leri et al., 2012;Özcelik and Koka, 2012;Seyyednejad et al., 2008;Shams and Schmidt, 2007). Various products from seed kernels and fruits of mahaleb tree have many uses as pleasing spice in patisseries mixed with flour for their special fragrance, home baking and candy industry (Özcelik and Koca, 2012). It has been used in folk medicine in various ailments as tonic for sensory organs, heart diseases, asthma, blood pressure, diabetes, swelling of stomach, relieving pains arising from liver, kidney swelling, anti-kidney stones, inflammation, oxidative stress diseases, gastrointestinal problem, diarrhea, and for scenting and preservation purposes (Gerardi et al., 2010;Oskoueian et al., 2012;Shams et al., 2007). These plants are of significant potential in therapeutic applications against human pathogens including bacteria, fungi, and viruses (Holetz et al., 2002;Perez, 2003;Syyednejad, 208). All methanol extracts from different parts of mahaleb including flowers, leaves, branches, fruits, fruit stalks, seed and seed coat showed antibacterial and antifungal activities (Özcelik and Koca, 2012). The ethanolic extracts of mahaleb had antibacterial activity against Proteus mirabilis, Bacillus anthracis, and Staphylococcus aureus (Seyyednejad et al., 2008).

Growth of Aspergilli Strains on Media Enriched with Myrrha Resin and Mahaleb Extract
The strains of A. flavus and A. parasiticus were grown on Potato Dextrose Agar (PDA) and incubated for 7 days at ambient temperature (25-32°C). Glass tubes of 5 mm in diameter were sterilized and used to cut several discs from each of the growing fungal colonies. Inoculum from the growing colonies were added to 250 flasks containing 200 ml of yeast malt broth with different concentrations of myrrh resin (0.0%, 1.0%, 1.25%, 2.5% and 3.25% w/v), and mahaleb extract (0.0%, 0.75%, 1.5%, 2.5%, and 3.5% w/v). Three replicates were incubated at 25-32 o C for 15 days. Other sets were kept to measure the dry weight of the fungal mycelia using Oven method.

Effect of the Extracts on Synthetic Aflatoxin B 1
Pure aflatoxin B 1 of 885 ppb concentration was prepared in 100 ml sterile distilled water. The highest concentrations of resin (3.25%) and mahaleb (3.5%) were separately added to flasks containing pure aflatoxin B 1 . As a control, flask containing aflatoxin B 1 was left without any extract. The flasks were incubated at 25-32 o C for 7 days and aflatoxin concentrations were assessed.

Extraction and Assay of Aflatoxin
Alfa Test-P Affinity method was used for aflatoxin extraction and detection as described by many authors and adopted in our previous study (El-Nagerabi et al., 2012). To the 200 ml fungal culture, 5g of sodium chloride in addition to 100 ml methanol:water (70:30 V/V) as extraction solution were added. To the filterate, 15 ml distilled water were added, mixed, filtered with glass microfilters. Ten ml from the diluted filtrate were passed via Afla-Test-P Affinity Column and the column was cleaned by 10 ml distilled water. The extracted aflatoxin was eluted with one ml methanol (HPLC grade) and one ml of AflaTest developer was added to elute in the cuvette, and vortexed. The aflatoxin concentration was measured by calibrated Vicam fluorometer (Series-4EX) (El-Nagerabi et al., 2016;Elshafie and Al-Shally, 1998).

Statistical Analysis
One-way ANOVA test (correlation coefficient) under SPSS software (version 11.0) was used to determine the variation between the effects of different concentrations of C. myrrha resin gum and P. mahaleb seed extract on aflatoxin inhibition-detoxification and fugal growth.

Conclusion
We screened the biological activities of different concentrations of C. myrrha resin and P. mahaleb seed extract on the growth and aflatoxin production by A. flavus (SQU21) and A. parasiticus (CBS921.7). These two plant extracts evidently reduce aflatoxin production and the fungal growth which may suggests the inhibitory effect to aflatoxin biochemical synthesis pathway. None of the two extracts detoxify pure aflatoxin B 1 as suggested by many researchers (Abulmajeed, 2011;Banno et al., 2006;El-Nagerabi et al., 2012, 2016Gupta et al., 2001;Langmead and Rampton 2006;Miller & Morris, 2004;Mothana et. al. 2011;Suhail et al. 2011). Therefore, toxicity of biologically active chemical components which reduce aflatoxin production needs more attention. This will build the data on their applications in food preservation industry and pharmaceutical activities.